title: Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability.
abstract: How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.
authors.affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Choy
authors.forename: Eileen
authors.initials: E
authors.lastname: O'Toole
authors.forename: Breanna M
authors.initials: BM
authors.lastname: Schuster
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Crisp
authors.forename: Tatiana S
authors.initials: TS
authors.lastname: Karpova
authors.forename: James G
authors.initials: JG
authors.lastname: McNally
authors.forename: Mark
authors.initials: M
authors.lastname: Winey
authors.forename: Melissa K
authors.initials: MK
authors.lastname: Gardner
authors.forename: Munira A
authors.initials: MA
authors.lastname: Basrai
mesh_terms: D020090:Chromosome Segregation; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D042822:Genomic Instability; D057895:Haploinsufficiency; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D022101:Microtubule-Organizing Center / Q000378:metabolism; D008870:Microtubules / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008941:Spindle Apparatus / Q000502:physiology; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
chemical_list: D003598:Cytoskeletal Proteins; D008869:Microtubule-Associated Proteins; C465662:SPC97 protein, S cerevisiae; C552429:SPC98 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C100764:TUB4 protein, S cerevisiae; D014404:Tubulin